Arnold E Ruoho - US grants

The goal of this research project is identification, isolation, and molecular characterization of the 8-adrenergic receptor. This will be accomplished by the use of specific highly radioactive photoaffinity labelling reagents which are derivations of potent beta-antagonists, such as propranolol and pindolol. Purification of the receptor will be attempted using alprenolol affinity columns. Detergent extracts of membranes containing receptor-beta-receptor antagonist (with biotinyl groups attached) will be passed over the columns to effect purification of the receptor. Other aspects of this proposal involve investigations on the involvement of sulfhydryl groups in the mechanism by which catecholamines activate adenylate cyclase. Distribution of the receptor on the surface of cells will be investigated using bifunctional ligands containing biotin and the protein, avidin, suitably derivated for microscope visualization.

This proposal involves the development and use of photoactive radioactive compounds which will be used to identify various domains within the beta- adrenergic receptor (betaAR), the GTP-binding regulatory proteins (G- protein), and adenylyl cyclase. We will develop novel antagonist and agonist radioiodinated photoactive compounds which are selective for the catechol portion of the beta2AR ligand binding site. These compounds will be used to photoaffinity label the ligand binding domain of the baculovirus-expressed Sf9 beta2-adrenergic receptor (rbeta2AR). Partial N-terminal or complete sequence will be obtained for [125I]photolabeled peptides using a variety of photoprobes to "map" the binding domain of the beta2AR. Novel radioiodinated forskolin photolabels with a potential for high insertion yield will be prepared and the forskolin binding site in adenylyl cyclases (types I, IV, and VI) will be identified. Adenylyl cyclase (I, V, VI) will be overexpressed in Sf9 cells and purified as a polyhistidine epitope-tagged enzyme. Intramolecular and intermolecular domains of Gs, Gi, Go, and Gt will be identified by use of radiolabel transfer from "tethered" molecules. This work will involve the preparation of novel radioactive NAD+ photoactivatable molecules and of sulfhydryl "tethered" photoactivatable molecules. Specific peptides derived from the sequence of the beta2AR, Gsalpha, Gtalpha will be derivatized to produce photoaffinity peptides (photopeptides) to identify interacting domains between beta2AR-Gs and Gs-beta2AR, between alphat and rhodopsin, and between alphat and cGMP phosphodiesterase. The location and intramolecular domains of the sulfhydryl and disulfides of the rbeta2AR and rhodopsin will be assessed by titration with sulfhydryl- specific reagents. Label transfer experiments will be performed to probe the intramolecular structure of the beta2AR. An assessment of the alpha- helical character of the beta2AR transmembrane 5 (TM 5) segment will be performed by use of the Nuclear Overhauser Effect (NOE) in NMR experiments. The approaches outlined in this proposal will be applicable to the study of intramolecular and intermolecular interactions between polypeptides in a variety of proteins. These experiments will increase our understanding of catecholamine beta-receptors which function to control autonomic functions such as heart rate, blood pressure, neuronal function, and metabolic states of liver, adipose, and muscle.

The strategy of this research proposal is based on the rationale that a basic understanding of the mechanism of action of cocaine both centrally and peripherally depends upon the biochemical characterization of the proteins which bind this drug. This proposal involves the development and use of novel radioiodinated photoaffinity probes derived from cocaine, from 2beta-carbomethoxy-3beta- (4-fluorophenyl)tropane (CFT), and from specific monoamine uptake inhibitors (i.e., imipramine, desimipramine). These probes will be used to photolabel monoamine transporters in rat striatal synaptosomes (dopamine), chromaffin cell membranes (NE), and platelet membranes (serotonin). Photoaffinity labeling of HeLa cell membranes which contain overexpressed dopamine and norepinephrine transporters will also be performed. Transporters which are labeled with these photoprobes will be identified by SDS-PAGE and pharmacologically characterized, as defined by the inhibition of labeling by specific monoamine uptake inhibitors (i.e., mazindol and GBR 12935 for the dopamine transporter, imipramine and fluoxetine for the serotonin transporter, and desipramine for the norepinephrine transporter). In the cases where two or more photoprobes (i.e., derivatives of cocaine, imipramine, and GBR 12935) label the same protein, peptide mapping will be performed to assess the identity of their binding sites. All photolabels will be characterized functionally as inhibitors of dopamine, norepinephrine, and serotonin transport using striatal synaptosomes, chromaffin cells, and platelets, respectively. HeLa cells which overexpress the dopamine and norepinephrine transporters will also be used in functional assays for cocaine photolabels. Cocaine affinity column resins will be synthesized. These resins will be chemically and functionally characterized and used towards purifying monoamine transporters from detergent extracts of HeLa membranes which contain overexpressed dopamine and norepinephrine transporters. A combination of biochemical techniques will be used for purification. Photolabeled peptides will be purified and sequenced to identify the cocaine binding site. The approaches outlined in this proposal will be used to characterize the molecular mechanism of action of cocaine towards a more rational approach in the effective treatment of cocaine abuse.

The sequestration of neurotransmitters, such as serotonin, norepinephrine, dopamine, and histamine into intracellular vesicles for subsequent release is a fundamental cellular event in neurons and secreting cells. Reduced or aberrant activity of the monoamine translocator of the synaptic vesicle may play a central role in Parkinson's Disease. Reserpine, used clinically for many years to control hypertension, causes the depletion of amines from the storage vesicles by inhibition of the monoamine translocator in the vesicle membrane. The regulation of uptake of monoamine neurotransmitters into storage vesicles may play an important role in affective psychological disorders related to responses has a paracrine action in the gut, and is a neurotransmitter in the central nervous system. The strategy of this proposal is based on the rationale that identification of the reserpine binding site on monoamine transporters will provide a basic understanding of the biochemical mechanism of action of monoamine sequestration into vesicles and the factors which regulate the activity of the translocator. The binding site for reserpine and reserpine-like compounds on monoamine vesicle translocators will be mapped. This work will be accomplished in four Specific Aims: (1) a series of novel radioiodinated and tritiated reserpine and tetrabenazine photoaffinity probes will be developed; (2) photolabels will be functionally assessed as inhibitors of monoamine uptake in chromaffin granules and in reconstituted vesicles containing pure recombinant baculovirus-Sf9-expressed translocators; (3) overexpression and large- scale purification of the synaptic vesicle transporter (SVAT), the chromaffin granule transporter (CGAT), and the mast cell histamine transporter (HIST) will be performed; purification strategies will be based on polyhistidine epitope tagging, lectin affinity chromatography, and ion exchange chromatography; (4) photolabeled peptides will be prepared and identified by combined strategies of chemical and tic cleavage, purification by HPLC, SDS-PAGE, and/or monoclonal and peptide- specific antibodies and N-terminal microsequencing. This research will provide a logical protein chemistry-based approach for assessment of the structure of the reserpine binding site of the vesicle monoamine transporter. This work will provide insight into the mechanism of action of the monoamine transporters and contribute to our understanding of how pharmacological and therapeutic strategies may be devised to treat Parkinsonism, cardiovascular disease, allergy, and disorders of the nervous system.

The focus of this proposal is to understand the molecular interactions which occur in the various domains of the receptor- G protein-effector coupling system through the use of multiple approaches. Novel antagonist and agonist radioiodinated photoactive compounds which are selective for the catechol portion of the b2AR ligand binding site will be developed. These compounds will be used to photoaffinity label the ligand binding domain of the baculovirus-expressed Sf9 b2-AR. Partial N-terminal or complete sequence will be obtained for [125I] photolabeled peptides using a variety of photoprobes to "map" the binding domain of the b2AR. The "exosite" on the b2AR, which is involved in the mechanism of action of long-acting b2 agonists (i.e., salmeterol), will be identified using novel salmeterol derivatives. Soluble cytoplasmic domains of adenylyl cyclase will be expressed in E. coli and purified. The forskolin, ATP, and inhibitory "p" site on the soluble adenylyl cyclase will be identified using photoactivatable compounds and purification of photolabeled peptides. Photolabeling experiments will also be performed on intact adenylyl cyclase, which will be overexpressed in Sf9 cells. Photoactivatable derivatives of specific domain-interacting peptides derived from the sequences of the b2AR and Gas will be utilized to identify interacting domains between b2AR and Gas, between Gas and adenylyl cyclase, and between rhodopsin and alpha transducin. The crystal structure of the catalytically active soluble IC1 and IIC2 adenylyl cyclase will be determined. The binding site domains for forskolin, nucleotides, and Gas will also be determined in the catalytically active soluble IC1 and IIC2 adenylyl cyclase by generating co-crystals. A component of this work involves expression and purification of large quantities of the IC1 domain and/or construction of catalytically active hybrid IC1. IIC2 molecules which are suitable for crystallography. These experiments will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine beta-receptors, which function to control autonomic functions, such as heart rate, blood pressure, and neuronal function and metabolic state of liver, adipose, and muscle.

animal tissue; virus; proteins; nucleic acids; human tissue; hormones; biomedical resource; biological products;

Ser204 and Ser207 on Transmembrane 5 (TM5) of the ?2 adrenergic receptor (?2AR) are critical in interactions with catecholamines and other ?2AR binding ligands. It is believed that the hydroxyls of these two amino acid residues form hydrogen bonds with ?2AR agonists and antagonists. In this research project, a recombinant ?2AR with Ser204 to Cys (S204C) or S207C will be constructed, expressed in sf9 cells of baculovirus expression system. The purified receptor will be radiolabeled with thiocatecholamine derivatives ([125I]-1-(1-oxy-3-hydroxy-4-thio-phenyl) -3-(N-iodo-tyramine)-2-propanol and ([125I]-1-(1-oxy-3-thio-4-hydroxy-phenyl)-3-(N-iodo-tyramine)-2-propanol which have a thio group attached to the catechol phenyl ring instead of a hydroxyl group. In addition, novel photoactivable radioactive fluorenone and benzophenone derivatives have been synthesized for the binding site probing. The photoradiolabeled ?2AR will be cleaved by proteases and the peptide mapping will be analyzed with polyclonal antibodies to TM5 using Western blotting techniques. By sequencing the protealytic peptides, the binding sites for those ligands can be identified by comparing the obtained sequence with the deduced amino acid sequence of the ?2AR from the cDNA.

This research covers areas of polyethylene structure/property relationships and catalyst mechanism studies based upon a detailed analysis of the polymners made by a catalyst. Co-monomer identification, quantitation and sequence distribution using Markovian statistics to fir NMR data is a large part of this work, however the samples run at NMRFAM will be aimed at quantitation of branch carbons in polyethylenes. The quantitation of branch carbons in polyethylene at levels as low as 1/100,000 total carbons is important to understand differences in the film properties of these polymers.

DESCRIPTION (Investigator's Abstract): The sequestration of neurotransmitters, such as serotonin, norepinephrine, dopamine, and histamine into intracellular vesicles for subsequent release is a fundamental cellular event in neurons and secreting cells. Reduced or aberrant activity of the monoamine translocator of the synaptic vesicle may play a central role in Parkinson's Disease. Reserpine, used clinically for many years to control hypertension, causes the depletion of amines from the storage vesicles by inhibition of the monoamine translocator in the vesicle membrane. The regulation of uptake of monoamine neurotransmitters into storage vesicles may play an important role in affective psychological disorders related to depression by altering levels of serotonin, norepinephrine, dopamine, or other neurotransmitters. The strategy of this proposal is based on the rationale that identification of the inhibitor, substrate, and proton translocation sites on monoamine transporters and VMAT-interacting proteins will provide a basic understanding of the biochemical mechanism of action of monoamine sequestration into vesicles and the factors which regulate the activity of the translocator. This work will be accomplished in four Specific Aims: (1) identification of the VMAT2 substrate binding site(s) using novel derivatives of amphetamine, dopamine, and the neurotoxin MPP+; (2) identification of the reserpine binding site(s) on VMAT2 using reconstituted pure VMAT2 in artificial liposomes and novel radioiodinated reserpine photolabels; (3) identification of the specific [14C]dicyclohexylcarbodiimide (DCCD)-reactive amino acid in VMAT2 which is involved in proton transport; novel cleavable DCCD-based photolabels to probe the molecular environment of the DCCD-reactive site will be prepared; (4) identification of proteins which interact with VMAT using multiple biochemical and molecular biology techniques. This research will provide a logical protein chemistry-based approach for assessment of the structure and regulation of the vesicle monoamine transporter. This work will provide insight into the mechanism of action of the monoamine transporters and contribute to our understanding of how pharmacological and therapeutic strategies may be devised to treat Parkinsonism, cardiovascular disease, allergy, and disorders of the nervous system.

DESCRIPTION (provided by applicant): The focus of the proposed research is to understand the molecular interactions which regulate G protein-coupled receptor (GPCR) signaling through the use of multiple approaches. This work will involve the following Specific Aims: (1) To continue experiments to characterize the "exosite" in the human b2AR using the newly developed I[125] iodoazidosalmeterol. This new photolabel is designed to covalently react with that portion of the b2AR, which interacts with the hydrophobic aryloxyalkyl tail which contributes to the long-acting property of salmeterol. Radiolabeled peptides will be generated from the photolabeled human b2AR, which is overexpressed in cultured HEK 293 cells. These peptides will be purified, N-terminal sequenced, and their position in the b2AR sequence determined. The precise derivatization sites will be determined by radiosequencing. (2) To identify interacting domains using "tethered molecules." Development and use of photoactivatable derivatives of specific domain-interacting peptides, which will allow identification of intermolecular domains, which interact between the b2AR and Gas and between rhodopsin and a transducin (at). Chimeric proteins consisting of bacteriorhodopsin (BR) and intracellular loops of rhodopsin and the b2AR will be co-crystallized with holotransducin (BR/rhodopsin) and with Gs (BR/b2AR). Emphasis will be placed on the crystal structure of BR/i-3 loop chimeras initially with the cognate G proteins. (3) To characterize the interaction of the cGMP phosphodiesterase gammasubunit (PDEgamma) with the a subunit of transducin in the absence and presence of RGS-9/Gb5. This Aim will involve the use of full-length PDEgamma photoaffinity labels containing benzophenones at various positions throughout the PDEgamma sequence. Using purified transducin and PDEgamma photolabels, crosslinked peptide sequences will be identified using mass spectroscopy and individual Gat residues identified using reversible benzophenone PDEgamma derivatives. Additional structural information will be obtained throught the use of NMR spectroscopy on the transducin bound form of PDEgamma. (4) To determine the role of synapse associated proteins (SAPs), in particular SAP97, in b2AR function. This Aim involves characterization of the interaction between SAPs and b2AR and identification of other partners, such as alpha-actinin, actin, and adenylyl cyclase type VI, in the signaling complex. This will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine b-receptors, which play a major role in the regulation of heart rate, blood pressure, heart failure, and other cardiovascular pathologies.

DESCRIPTION (provided by applicant): The focus of the proposed research is to understand the molecular interactions which regulate G protein-coupled receptor (GPCR) signaling through the use of multiple approaches. This work will involve the following Specific Aims: (1) To continue experiments to characterize the "exosite" in the human b2AR using the newly developed I[125] iodoazidosalmeterol. This new photolabel is designed to covalently react with that portion of the b2AR, which interacts with the hydrophobic aryloxyalkyl tail which contributes to the long-acting property of salmeterol. Radiolabeled peptides will be generated from the photolabeled human b2AR, which is overexpressed in cultured HEK 293 cells. These peptides will be purified, N-terminal sequenced, and their position in the b2AR sequence determined. The precise derivatization sites will be determined by radiosequencing. (2) To identify interacting domains using "tethered molecules." Development and use of photoactivatable derivatives of specific domain-interacting peptides, which will allow identification of intermolecular domains, which interact between the b2AR and Gas and between rhodopsin and a transducin (at). Chimeric proteins consisting of bacteriorhodopsin (BR) and intracellular loops of rhodopsin and the b2AR will be co-crystallized with holotransducin (BR/rhodopsin) and with Gs (BR/b2AR). Emphasis will be placed on the crystal structure of BR/i-3 loop chimeras initially with the cognate G proteins. (3) To characterize the interaction of the cGMP phosphodiesterase gammasubunit (PDEgamma) with the a subunit of transducin in the absence and presence of RGS-9/Gb5. This Aim will involve the use of full-length PDEgamma photoaffinity labels containing benzophenones at various positions throughout the PDEgamma sequence. Using purified transducin and PDEgamma photolabels, crosslinked peptide sequences will be identified using mass spectroscopy and individual Gat residues identified using reversible benzophenone PDEgamma derivatives. Additional structural information will be obtained throught the use of NMR spectroscopy on the transducin bound form of PDEgamma. (4) To determine the role of synapse associated proteins (SAPs), in particular SAP97, in b2AR function. This Aim involves characterization of the interaction between SAPs and b2AR and identification of other partners, such as alpha-actinin, actin, and adenylyl cyclase type VI, in the signaling complex. This will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine b-receptors, which play a major role in the regulation of heart rate, blood pressure, heart failure, and other cardiovascular pathologies.

DESCRIPTION (provided by applicant): The strategy of this proposal is based on the rationale that identification of the inhibitor, substrate, proton translocation, and functionally relevant phosphorylation sites on monoamine transporters (VMAT2) will provide a basic understanding of the mechanism of action of monoamine sequestration into vesicles and the factors which regulate transporter activity. This work will be accomplished in three Specific Aims: (1) Identification of the reserpine binding site(s) on VMAT2. Novel reserpine photoaffinity labels will be synthesized and characterized, and photo-labelled peptides will be identified in order to map the reserpine binding site; (2) Identification of the substrate transport channel. This aim will involve the use of several approaches, including radioactive photo-activatable substrate analogs to covalently derivatize the substrate binding site on VMAT2; site-specific derivatization of VMAT2 at engineered cysteine residues with the cysteine-reactive reagents, methanethiosulfonate ethyl amine (MTSEA), and MTS-ethyltrimethylammonium (MTSET); and site-directed mutagenesis of potential residues lining the channel; (3) Determination of the functional role of two highly charged regions of VMAT2. This aim will involve the use of biochemical and genetic (site-directed mutagenesis) approaches to determine the role of phosphorylation of the N-terminus of VMAT2 on transporter function and the intracellular distribution/oligomeric state of the transporter. Reduced or aberrant activity of the monoamine transporter of the synaptic vesicles in dopaminergic neurons of the substantia nigra through either direct or indirect actions of toxicants (e.g., MPP+, insecticides) and genetically altered neuronally expressed proteins may play a central role in Parkinson's Disease. The regulation of uptake of monoamine neurotransmitters into storage vesicles may also play an important role in affective psychological disorders related to depression by altering levels of serotonin, norepinephrine, dopamine, or other neurotransmitters. This work will provide insight into the mechanism of action of the monoamine transporters and contribute to our understanding of how pharmacological and therapeutic strategies may be devised to treat Parkinsonism or other disorders of the nervous system.

[unreadable] DESCRIPTION (provided by applicant): Sigma receptors are unique "non-opioid" receptors that are found in the mammalian central nervous system and peripheral organs. The function of the sigma receptor is unknown, but it is believed to mediate the immunosuppressant, antipsychotic, and neuroprotective effects of drugs, such as haloperidol, ditolylguanidine (DTG), pentazocine, and cocaine. Several observations indicate potential therapeutic uses of sigma ligands. Some atypical neuroleptics (e.g., haloperidol) have high affinity for the sigma receptor, which has led to the possibility of creating a new class of antipsychotic drugs, which are devoid of dopaminergic activity and can bind selectively to the sigma receptor. A genetic linkage has been reported for the sigma receptor and schizophrenia. Selective sigma ligands can block the behavioral and toxic functions of cocaine, and cocaine can also serve as a sigma ligand with reasonable affinity. These observations raise the possibility that the sigma receptor may be a target for the treatment of cocaine-related responses. The potent immunosuppressant SR31747A, which binds to the sigma receptor, exhibits immunosuppressive properties and antiproliferative activity in mouse and human T lymphocytes. These observations may explain the immunosuppressant properties of cocaine and other sigma ligands and lead to a new generation of immunosuppressants. A number of studies have shown that sigma receptor ligands modulate ion channels in the plasma membrane to regulate excitability. Target channels are general voltage-gated K+ channels, and the modulation entails an inhibition of the current elicited by positive voltage steps. The focus of this research proposal is to characterize the structure of the sigma1 receptor binding site and the manner by which the sigma1 receptor modulates K+ channels. Three areas of focus will be investigated: (1) synthesis and characterization of novel high affinity sigma receptor agonist and antagonist photo affinity labels; (2) mapping the ligand binding site(s) of the sigma1 receptor; and (3) determination of the properties of the sigma1 receptor interaction with Kvl.4 potassium channels and other protein partners. These experiments will be performed through the combined use of photoactivatable molecules, electrophysiology, and recombinant and fusion proteins.

[unreadable] DESCRIPTION (provided by applicant): Sigma receptors are unique "non-opioid" receptors that are found in the mammalian central nervous system and peripheral organs. The function of the sigma receptor is unknown, but it is believed to mediate the immunosuppressant, antipsychotic, and neuroprotective effects of drugs, such as haloperidol, ditolylguanidine (DTG), pentazocine, and cocaine. Several observations indicate potential therapeutic uses of sigma ligands. Some atypical neuroleptics (e.g., haloperidol) have high affinity for the sigma receptor, which has led to the possibility of creating a new class of antipsychotic drugs, which are devoid of dopaminergic activity and can bind selectively to the sigma receptor. A genetic linkage has been reported for the sigma receptor and schizophrenia. Selective sigma ligands can block the behavioral and toxic functions of cocaine, and cocaine can also serve as a sigma ligand with reasonable affinity. These observations raise the possibility that the sigma receptor may be a target for the treatment of cocaine-related responses. The potent immunosuppressant SR31747A, which binds to the sigma receptor, exhibits immunosuppressive properties and antiproliferative activity in mouse and human T lymphocytes. These observations may explain the immunosuppressant properties of cocaine and other sigma ligands and lead to a new generation of immunosuppressants. A number of studies have shown that sigma receptor ligands modulate ion channels in the plasma membrane to regulate excitability. Target channels are general voltage-gated K+ channels, and the modulation entails an inhibition of the current elicited by positive voltage steps. The focus of this research proposal is to characterize the structure of the sigma1 receptor binding site and the manner by which the sigma1 receptor modulates K+ channels. Three areas of focus will be investigated: (1) synthesis and characterization of novel high affinity sigma receptor agonist and antagonist photo affinity labels; (2) mapping the ligand binding site(s) of the sigma1 receptor; and (3) determination of the properties of the sigma1 receptor interaction with Kvl.4 potassium channels and other protein partners. These experiments will be performed through the combined use of photoactivatable molecules, electrophysiology, and recombinant and fusion proteins.

[unreadable] DESCRIPTION (provided by applicant): Sigma receptors are unique "non-opioid" receptors that are found in the mammalian central nervous system and peripheral organs. The function of the sigma receptor is unknown, but it is believed to mediate the immunosuppressant, antipsychotic, and neuroprotective effects of drugs, such as haloperidol, ditolylguanidine (DTG), pentazocine, and cocaine. Several observations indicate potential therapeutic uses of sigma ligands. Some atypical neuroleptics (e.g., haloperidol) have high affinity for the sigma receptor, which has led to the possibility of creating a new class of antipsychotic drugs, which are devoid of dopaminergic activity and can bind selectively to the sigma receptor. A genetic linkage has been reported for the sigma receptor and schizophrenia. Selective sigma ligands can block the behavioral and toxic functions of cocaine, and cocaine can also serve as a sigma ligand with reasonable affinity. These observations raise the possibility that the sigma receptor may be a target for the treatment of cocaine-related responses. The potent immunosuppressant SR31747A, which binds to the sigma receptor, exhibits immunosuppressive properties and antiproliferative activity in mouse and human T lymphocytes. These observations may explain the immunosuppressant properties of cocaine and other sigma ligands and lead to a new generation of immunosuppressants. A number of studies have shown that sigma receptor ligands modulate ion channels in the plasma membrane to regulate excitability. Target channels are general voltage-gated K+ channels, and the modulation entails an inhibition of the current elicited by positive voltage steps. The focus of this research proposal is to characterize the structure of the sigma1 receptor binding site and the manner by which the sigma1 receptor modulates K+ channels. Three areas of focus will be investigated: (1) synthesis and characterization of novel high affinity sigma receptor agonist and antagonist photo affinity labels; (2) mapping the ligand binding site(s) of the sigma1 receptor; and (3) determination of the properties of the sigma1 receptor interaction with Kvl.4 potassium channels and other protein partners. These experiments will be performed through the combined use of photoactivatable molecules, electrophysiology, and recombinant and fusion proteins.

DESCRIPTION (provided by applicant): The photoreceptor PDE6 gamma subunit (Pg) is a critical regulatory molecule in rod and core photoreceptors cells and is also an important player in normal retinal development. The focus of the proposed research is to understand the molecular interactions of Pg, which regulates G protein-coupled receptor (GPCR) signaling in the visual system. This work will involve two Specific Aims: (1) To identify the interaction interfaces of the full-length PDEg molecule and the transducin alpha subunit (Gat) in the "GTP signaling state," in the "GDP-AIF4-transition state," and in the "RGS9/Gb5 transition state" complex. These experiments will be performed utilizing two complimentary approaches: (a) A systematic photolabel transfer to native and recombinant Gat from full-length PDEg photoprobes will be utilized to define the PDEg interaction sites on Gat complexed with GatGTPgS, GatGDP-AIF4- and in the full-length GatGDP-AIF4- /RGS9/Gb5 transition state complex using purified proteins followed by proteolytic cleavage and mass spectrometry analysis;(b) The solution structures of PDEg complexed with GatGTPgS, GatGDP-AIF4-, and recombinant Gat (Chi-8HN) will be determined by NMR spectroscopy utilizing 2H/13C/15N labeled proteins. (2) To identify the interaction surfaces between the PDE6 gamma subunit (PDEg), cyclic GMP and the PDE6 catalytic heterodimer subunits (PDEg). These experiments will be performed utilizing the following approaches: (a) The interaction site(s) on PDEg GAF domains that interact with the PDEg polycationic region will be identified through systematic photolabel transfer from PDEg photoprobes to PDEg, followed by proteolytic cleavage and mass spectrometry analysis;(b) The nature and modulation by PDEg of the cyclic GMP (cGMP) binding to the allosteric sites on the PDEg heterodimer will be elucidated utilizing non-radiolabeled and radiolabeled cGMP photolabeling experiments followed by proteolytic cleavage, SDS-PAGE and mass spectrometry analysis;(c) The NMR solution structure of the full-length PDEg complexed with the catalytic core of a PDE5/6 chimera will be determined using 2H/13C/15N labeled proteins. This work will provide important new knowledge in understanding regulation of GPCR-mediated signal transduction mechanisms in the visual system and the molecular interactions that are abrogated, leading to various forms of stationary night blindness in humans.

DESCRIPTION (provided by applicant): The Sigma-2 receptor is a unique protein found in most mammalian tissues. Sigma-2 receptors play an important role in growth regulation in normal cells and tumor cells of neuronal and non neuronal origin. The Sigma-2 receptor has not been purified or cloned. This grant proposal is directed at purification and cloning of the Sigma-2 receptor. This goal will be accomplished through two alternate and complementary approaches. Specific Aim 1 is entitled: Purification, Identification and Cloning of the Sigma-2 receptor using a "Click Chemistry" approach. Specific Aim 2 is entitled: Identification and cloning of the Sigma-2 receptor using an In Silico approach. Characterization of the Sigma-2 receptor is the initial step leading to a basic understanding of the role of the Sigma-2 receptor in regulation of neuronal and non-neuronal cell function and for potential therapeutic strategies to treat drugs of abuse and/or aberrant cell growth. . PUBLIC HEALTH RELEVANCE: The sigma-2 receptor is a mammalian protein in the central nervous system and in peripheral organs that may be involved in drug addiction. Sigma-2 receptors play an important role in growth regulation in normal cells and in tumor cells of different origins. Despite the presence of numerous molecular biology tools, the sigma-2 receptor has managed to evade all cloning attempts to date. This project is directed at purification and identification of the sigma-2 receptor.

DESCRIPTION (provided by applicant): The Sigma-1 receptor (S1R) is a 26 kDa protein found in portions of the endoplasmic reticulum (ER) of cells. In the central nervous system the highest levels of the S1R (CNS) are found in the postsynaptic portions of cholinergic postsynaptic densities (C-terminals) of spinal cord motoneurons. The primary objective of this proposal is to evaluate the role of S1R in regulating the excitability of motoneurons. We hypothesize that the S1R plays a direct role in regulating motoneuron excitability by activation of SK and/or Kv2.1 potassium channels in C-terminals, the effect of which is to increase the amplitude of the afterhyperpolarization potential (AHP) and thereby to reduce the frequency and duration of action potential firing. We hypothesize that activation of S1R can increase longevity of ALS mice by acting as a neuromodulatory brake on motoneuron hyperexcitability and thereby to decrease intracellular stress. We will accomplish the goals of this proposal through the following specific aims: Specific Aim 1: To determine whether S1R modulates motoneuron excitability (Electrophysiology) and influences the amplitude of electrical activity of the gastrocnemius muscle. (Motor activity). Specific Aim 2: To establish that activation of S1R can retard the progression of motoneuron degeneration in a mouse model of ALS (Pathology) by a) assessing survival of a new mouse model SOD1 ALS/S1R KO compared to the SOD1 ALS/S1R WT mice and b) assessing survival of the SOD1 ALS/S1R KO and the SOD1 ALS/S1R WT mice in the presence and absence of agonists of the S1R. Targeting S1R can potentially establish a new therapeutic treatment for ALS. PUBLIC HEALTH RELEVANCE: The hypothesis in this proposal is that the Sigma-1 receptor acts as a brake on neuronal excitability. With a reduction in neuronal excitability motoneurons experience less cellular damage and thereby remain functional for a longer period during the development of Amyotrophic Lateral Sclerosis (Lou Gehrig's disease). We expect that activating the Sigma-1 receptor with specific drugs will further reduce cellular damage and lead to new treatment strategies for Lou Gehrig's disease.

DESCRIPTION (provided by applicant): Neuropathic pain resulting from chronic inflammation and injury to the nervous system is particularly difficult to treat with the currently available drg armamentarium and is a highly problematic unsolved clinical problem. Development of a novel treatment for neuropathic pain is highly significant. Many signaling molecules are likely to partake in the manifestation of neuropathic pain. The Sigma 1 receptor (S1R) is a two-transmembrane mostly endoplasmic reticulum resident protein with many roles in fundamental cellular processes. One of the most robust phenotypes described for the S1R knockout mice is the suppression of neuropathic pain. Intrathecal administration of S1R antagonists mirrors this anti-neuropathic phenotype of the knockout indicating the importance of a spinal segmental level expression of S1R in mediating neuropathic pain. Based on the focused expression of S1R in the ventral horn in the spinal cord proper, we believe S1R in the dorsal root ganglion (DRG) is the key anatomical site of action. We propose a novel concept of the S1R as a master regulator of pronocicptive protein trafficking in the DRG as a mechanism underlying the anti- neuropathic phenotype observed in mice with pharmacological or genetic inhibition of this protein. Preliminary data documents protein:protein interaction between S1R and several pronociceptive proteins including the substance P receptor (NK1R), muscarinic M1 receptor (M1R), and the NR1 subunit of the NMDA receptor all sharing the ability to increase intracellular calcium signaling. Co-expression of S1R increases the plasma membrane expression of NK1R in these cells with a consequent increase in intracellular calcium signaling. We will test the highly innovative working hypothesis that: S1R protein upregulation and modulation of NK1R, M1R, and NMDAR in the dorsal root ganglion (DRG) mediates neuropathic pain. The proposal incorporates state-of-art techniques including a selective in vivo transduction and knockdown of S1R in the DRG by adenoassociated virus 2/8, extensive use of co-immunoprecipitation and novel molecular constructs to decipher the protein domains responsible for the interaction between S1R and the client proteins, and finally examines a gene therapy for neuropathic pain targeting S1R interaction with client proteins.